Multi-drug resistance has become an increasingly important cause of mortality and morbidity in humans. It is a pressing and continual need to discover new anti-infective agents. Bioactive natural products are a major source of anti-infective drugs. Traditionally, soil bacteria, especially the Gram-positive Streptomyces, have been the primary source for bioactive natural products. However, many ubiquitous inhabitants of soil and water, such as the Gram-negative gliding bacteria Lysobacter, remain largely unexplored, even though they are prolific producers of natural products. This proposal describes a research plan to discover and evaluate new anti-infectives with novel structures and potent activities from the new sources. The focus is on deciphering their biosynthetic and regulatory mechanisms. Specifically, two groups of bioactive natural products, HSAF and WAP-8294A, will be investigated. HSAF (dihydromaltophilin) is a polycyclic tetramate macrolactam (PTM) and represents a novel type of antifungal compounds with new chemistry and new mode of action. To develop HSAF as a new type of antifungal antibiotics, it is important to understand the biosynthetic mechanism for the novel chemical structure. The HSAF biosynthetic gene cluster contains only a single-module polyketide synthase-nonribosomal peptide synthetase (PKS/NRPS), flanked by a cascade of six redox enzymes, although HSAF biosynthesis apparently requires two separate hexaketide chains that are linked together by one amino acid (ornithine) via two amide bonds. This system represents an unprecedented iterative PKS/NRPS hybrid. The goal here is to illustrate the reactions catalyzed by the redox enzymes-modulated iterative PKS/NRPS, which lead to the distinct PTM structure. WAP-8294A are a complex of at least 20 cyclic lipodepsipeptides with a potent activity against Methicillin-Resistant Staphylococcus aureus (MRSA, ED5014 times more active than vancomycin, a last resort antibiotic). Due to a very low yield, only the major component of the complex, WAP-8294A2, is in clinical studies (by aRigen Pharmaceuticals). Although the compounds were first isolated nearly 15 years ago and one of them is in clinical studies, their biosynthetic genes were just identified in 2011. The goal here is to determine the molecular mechanisms for WAP biosynthesis and regulation, particularly the mechanism for fatty acid incorporation that leads to structural diversity. The understanding of the biosynthetic and regulatory mechanisms for HSAF and WAP-8294A is an essential step toward rational biosynthetic engineering, productivity improvement and structure-activity optimization of these two groups of very promising bioactive natural products. In addition, because Lysobacter are prolific natural product producers that have not been exploited, this research will open a new direction to discovering new anti-infective drugs.